Nuclear envelope localization of LEMD2 is developmentally dynamic and lamin A/C dependent yet insufficient for heterochromatin tethering.

Peripheral heterochromatin in mammalian nuclei is tethered to the nuclear envelope by at least two mechanisms here referred to as the A- and B-tethers. The A-tether includes lamins A/C and additional unknown components presumably INM protein(s) interacting with both lamins A/C and chromatin. The B-tether includes the inner nuclear membrane (INM) protein Lamin B-receptor, which binds B-type lamins and chromatin. Generally, at least one of the tethers is always present in the nuclear envelope of mammalian cells. Deletion of both causes the loss of peripheral heterochromatin and consequently inversion of the entire nuclear architecture, with this occurring naturally in rod photoreceptors of nocturnal mammals. The tethers are differentially utilized during development, regulate gene expression in opposite manners, and play an important role during cell differentiation. Here we aimed to identify the unknown chromatin binding component(s) of the A-tether. We analyzed 10 mouse tissues by immunostaining with antibodies against 7 INM proteins and found that every cell type has specific, although differentially and developmentally regulated, sets of these proteins. In particular, we found that INM protein LEMD2 is concomitantly expressed with A-type lamins in various cell types but is lacking in inverted nuclei of rod cells. Truncation or deletion of Lmna resulted in the downregulation and mislocalization of LEMD2, suggesting that the two proteins interact and pointing at LEMD2 as a potential chromatin binding mediator of the A-tether. Using nuclei of mouse rods as an experimental model lacking peripheral heterochromatin, we expressed a LEMD2 transgene alone or in combination with lamin C in these cells and observed no restoration of peripheral heterochromatin in either case. We conclude that in contrary to the B-tether, the A-tether has a more intricate composition and consists of multiple components that presumably vary, at differing degrees of redundancy, between cell types and differentiation stages.

LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation.

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Offspring after embryo-preserving biopsy of the embryoblast with standard ICSI equipment in mouse blastocysts.

BACKGROUND/AIM: Obtaining human embryonic stem cell lines has so far involved destroying the embryos. This has given rise to ethical concerns and is not permitted in most countries. This investigation tested whether removing multiple cells from blastocysts might allow continued embryonic development. MATERIALS AND METHODS: A total of 40 blastocysts from a black mouse strain were biopsied. The mouse blastocysts were fixed with a holding pipette. The zona pellucida and trophectoderm layer were penetrated with an injection pipette, and cells from the inner cell mass (ICM) were aspirated. The pipette was removed and the ICM cells were transferred into a medium. RESULTS: The blastocysts collapsed after pipette removal and were allowed to regenerate for 6 h. Twenty-four blastocysts recovered, expanded and were implanted into four white surrogate mothers. One surrogate mother gave birth to two black pups. CONCLUSION: This experiment demonstrates that nondestructive blastocyst biopsy from the ICM is possible in mice.

A novel approach to selectively target neuronal subpopulations reveals genetic pathways that regulate tangential migration in the vertebrate hindbrain.

Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications.

Evidence for an evolutionary conserved role of homothorax/Meis1/2 during vertebrate retina development.

During eye development in D. melanogaster, the TALE-homeodomain protein Homothorax (Hth) is expressed by progenitor cells ahead of the neurogenic wave front, promotes rapid proliferation of these cells and is downregulated before cells exit the cell cycle and differentiate. Here, we present evidence that hth function is partially conserved in vertebrates. Retinal progenitor cells (RPCs) in chicks and mice express two Hth-related proteins, Meis1 and Meis2 (Mrg1), in species-specific temporal sequences. Meis1 marks RPCs throughout the period of neurogenesis in the retina, whereas Meis2 is specific for RPCs prior to the onset of retinal differentiation. Transfection of Meis-inactivating constructs impaired RPC proliferation and led to microphthalmia. RNA-interference-mediated knock-down of expression indicated that progenitor cells expressing Meis1 together with Meis2 proliferate more rapidly than cells expressing Meis1 alone. Transfection of Meis-inactivating constructs reduced the expression of cyclin D1 (Ccnd1) in the eye primordium and co-transfection of cyclin D1 partially rescued RPC proliferation. Collectively, these results suggest that (1) Meis1 and Meis2, similar to hth, maintain retinal progenitor cells in a rapidly proliferating state; (2) they control the expression of some ocular-determination genes and components of the cell cycle machinery; and (3) together with the species-specific differences in Meis1/Meis2 expression, combinatorial expression of Meis family proteins might be a candidate mechanism for the differential regulation of eye growth among vertebrate species.

Isolation and expression pattern of three mouse homologues of chick Rgm.

Growth cones of developing neurons are guided to their targets by attractive and repulsive cues. We have isolated three mouse homologues of the chick 'repulsive guidance molecule', Rgm [Nature 419 (2002) 392], and characterized their expression patterns. RgmA and RgmB are predominantly expressed in the developing and adult central nervous system in distinct patterns with little overlap. The third member of the Rgm gene family, RgmC, shows exclusive expression in the muscle cell lineage. From sequence and expression data, we suggest RgmA to be the mouse orthologue of chick Rgm.

Cloning of three mouse Unc5 genes and their expression patterns at mid-gestation.

The Caenorhabditis elegans gene unc-5 and it's vertebrate homologues are Netrin receptors. In this study, I report the cloning of three mouse Unc5 family members, namely, Unc5h1, Unc5h2 and Unc5h4. Furthermore, a comparative expression analysis is presented with Unc5h3, deleted in colorectal cancer and Netrin-1. Transcript distribution is studied during early eye development, mammary bud formation, vascularisation, and limb development. The most widely expressed Unc5 family member is Unc5h2 and it's mRNA is observed during early blood vessel formation, in the semicircular canal and in a dorsal to ventral gradient in the retina. Unc5h1 expression is restricted to the central nervous system, whereas, sites of Unc5h4 expression are in the developing limb and mammary gland.